Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

Point mutations at threonine-301 modify substrate specificity of rabbit liver microsomal cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase)

Thr-301 of cytochrome P-450 (laurate (omega-1)-hydroxylase) was replaced by Ser, Val, Ile, or Asn via site-directed mutagenesis. The Ser-, Val-, and Asn-mutants had lower laurate (omega-1)-hydroxylase activities than the wild-type P-450. The mutation to Ser did not affect caprate (omega-1)-hydroxylase activity and rather increased caprate omega-hydroxylase activity, but the Val- and Asn-mutants could not hydroxylate caprate. The Ile-mutant was devoid of the hydroxylase activities. The mutation also led to changes in the affinities for the fatty acids and exogenous ligands. Replacement of Thr-301 of cytochrome P-450 (testosterone 16 alpha-hydroxylase) by Ser or Val also affected the activities toward testosterone and progesterone in different ways. These findings indicate that residue 301 of the P-450s plays an important role in determining their substrate specificities.     

Spectral quality during pod development affects omega-6 desaturase activity in soybean seed endoplasmic reticulum

Polyunsaturated fatty acids (i.e. linoleic acid [18:2], linolenic acid [18:3]) in triacylglycerols (TAG) of soybean seeds increase more during reproductive development under simulated shadelight: i.e., light with reduced blue and/or increased far-red (Britz and Cavins 1993). Elevation of 18:2 and 18:3 is matched by corresponding reduction of oleic acid (18:1), consistent with observations that total seed oil remains constant. We therefore tested the hypothesis that spectral quality affects the activity of the omega-6 and/or omega-3 desaturases involved in TAG biosynthesis. Membranes were isolated from developing soybean cotyledons 24–31 days after flowering. Separate fractions, enriched for chloroplasts and endoplasmic reticulum (ER) respectively, were obtained by sucrose gradient centrifugation and incubated in an in vitro desaturase assay system containing ¹⁴C-18:1–CoA at room temperature. Omega-6 and omega-3 desaturase activity was calculated from the rate of formation of ¹⁴C-18:2 and ¹⁴C-18:3. Radioactive 18:2 and 18:3 were recovered only from phosphatidylcholine (PC) in both ER and chloroplast membranes, consistent with membrane-bound desaturases with specificity towards PC. The specific activity of omega-6 desaturase was high in ER membranes from seeds matured under light sources that promote a canopy shade response, but was reduced in membranes from seeds matured under lamps (high blue and low far-red emission) previously shown to reduce the level of 18:2 in seed oil by 50%. Chloroplast membranes possessed both omega-6 and omega-3 desaturases. Light appeared to have little or no effect on the activity of chloroplast desaturases.     

Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium.

The fatty acid (omega-2) hydroxylase from Bacillus megaterium ATCC 14581 was examined with respect to some general enzymatic properties attributed to an intact complex isolated in a partially purified state. Hydroxylase specific activity was found to increase with increasing protein concentration in a manner consistent with a reversible association of the components in the complex. There was a substantial kinetic lag phase for palmitate hydroxylation which was abolished by a substrate preincubation in the absence of NADPH. The substrate bound and presumably activated the hydroxylase complex without the formation of a substrate-derived intermediated. The oxidation of NADPH and the hydroxylation of palmitate were found to occur in a one to one molar ration, independent of the protein concentration. Finally, a cytochrome P-450 component of the complex was identified on the basis of its CO-binding difference spectrum. It appears, that this cytochrome P-450 component is not identical to P-450 meg of the steroid hydroxylase system of B. megaterium ATCC 13368, since progesterone, an active substrate for the latter, is not hydroxylated by the preparation from B. megaterium ATCC 14581.     

Preventive effects of omega-3 and omega-6 Fatty acids on peroxide mediated oxidative stress responses in primary human trabecular meshwork cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H₂O₂, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H₂O₂ further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H₂O₂ stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H₂O₂ mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H₂O₂ induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.     

omega-1 and omega-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6

Incubation of prostaglandin E1 (PGE1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at omega-2, omega-1, and omega carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the omega-1 and omega-2 hydroxylation of PGE1 and PGE2. Additionally, a small amount of product with chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b5 into the reconstituted system did not enhance the rate of PGE1 hydroxylation and had no effect on the ratio of products formed. The Km value for the omega-1 and omega-2 hydroxylation of PGE1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 microM; the Vmax's (nmol product min-1 nmol P-450-1) were 2.1 and 1.1 for the omega-1 and omega-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the omega-2 hydroxylation of PGEs observed in rabbit liver microsomes.     

Agonistic approach of omega-3, omega-6 and its metabolites with BDNF: An in-silico study

Brain derived neurotrophic factor (BDNF) is a member of neurotrophic family of growth factors, mainly found in the hippocampus and cerebral cortex of brain. Studies have shown that there is a link between BDNF and cognitive dysfunction, as well as there is a relationship between the PUFAs intake and their effect on BDNF production. Intake of PUFAs, mainly omega-3 and omega-6 has show increase in production of BDNF in brain. In our study we performed docking studies on PUFAs and their metabolites with BDNF using MVD (Molegro Virtual Docker), this has shown that the metabolites of the PUFAs mainly LXA_4, NPD1, HDHA have shown more binding affinity towards BDNF. These metabolites of PUFAs are responsible for modulation of BDNF activity.     

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.     

Modulation of native T-type calcium channels by omega-3 fatty acids

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.     

Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity

cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells. The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase. When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.     

Association of the FADS2 gene with omega-6 and omega-3 PUFA Concentration in the egg yolk of Japanese quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low omega-6:omega-3 PUFA ratio (h2 = 0.36-0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the omega-6 and omega-3 PUFAs (P < 0.05). No effects of the other SNPs were found - indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to omega-6 and omega-3 PUFA concentration in the egg yolk.     

Regiospecific hydroxylation of lauric acid at the (omega-1) position by hepatic and kidney microsomal cytochromes P-450 from rainbow trout

Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout.     

Protein kinase inhibition by omega-3 fatty acids

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.     

Influence of omega-3 Fatty Acid status on the way rats adapt to chronic restraint stress

Omega-3 fatty acids are important for several neuronal and cognitive functions. Altered omega-3 fatty acid status has been implicated in reduced resistance to stress and mood disorders. We therefore evaluated the effects of repeated restraint stress (6 h/day for 21 days) on adult rats fed omega-3 deficient, control or omega-3 enriched diets from conception. We measured body weight, plasma corticosterone and hippocampus glucocorticoid receptors and correlated these data with emotional and depression-like behaviour assessed by their open-field (OF) activity, anxiety in the elevated-plus maze (EPM), the sucrose preference test and the startle response. We also determined their plasma and brain membrane lipid profiles by gas chromatography. Repeated restraint stress caused rats fed a control diet to lose weight. Their plasma corticosterone increased and they showed moderate behavioural changes, with increases only in grooming (OF test) and entries into the open arms (EPM). Rats fed the omega-3 enriched diet had a lower stress-induced weight loss and plasma corticosterone peak, and reduced grooming. Rats chronically lacking omega-3 fatty acid exhibited an increased startle response, a stress-induced decrease in locomotor activity and exaggerated grooming. The brain omega-3 fatty acids increased as the dietary omega-3 fatty acids increased; diets containing preformed long-chain omega-3 fatty acid were better than diets containing the precursor alpha-linolenic acid. However, the restraint stress reduced the amounts of omega-3 incorporated. These data showed that the response to chronic restraint stress was modulated by the omega-3 fatty acid supply, a dietary deficiency was deleterious while enrichment protecting against stress.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.     

Effect of saturated, omega-3 and omega-6 polyunsaturated fatty acids on myocardial infarction

Dietary fatty acids have cholesterol lowering, antiatherogenic, and antiarrhythmic properties that decrease the risk of myocardial infarction (MI). This study was designed to study the effects of various oils rich in either polyunsaturated (omega-3 or omega-6) fatty acids (PUFA) or saturated fatty acids (SFA) on the severity of experimentally induced MI. Male albino Sprague-Dawley rats (100-150 g; n = 20) were fed diets enriched with fish oil (omega-3 PUFA), peanut oil (omega-6 PUFA), or coconut oil (SFA) for 60 days. Experimental MI was induced with isoproterenol. Mortality rates; serum enzymes aspartate amino transferase; alanine amino transferase; creatine phosphokinase (CPK); lipid profiles in serum, myocardium, and aorta; peroxide levels in heart and aorta; activities of catalase and superoxide dismutase; and levels of glutathione were measured. The results demonstrated that mortality rate, CPK levels, myocardial lipid peroxides, and glutathione levels were decreased in the omega-3 PUFA treated group. Maximum increase in parameters indicative of myocardial damage was seen in the coconut oil group. These findings suggest that dietary omega-3 PUFA offers maximum protection in experimentally induced MI in comparison to omega-6 PUFA and SFA enriched diets. SFA was found to have the least protective effect.     

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.     

Delay of preterm delivery in sheep by omega-3 long-chain polyunsaturates

A positive correlation has been shown between dietary intake of long-chain omega-3 fatty acids in late pregnancy and gestation length in pregnant women and experimental animals. To determine whether omega-3 fatty acids have an effect on preterm labor in sheep, a fish oil concentrate emulsion was continuously infused to six pregnant ewes from 124 days gestational age. At 125 days, betamethasone was administered to the fetus to produce preterm labor. Both the onset of labor and the time of delivery were delayed by the fish oil emulsion. Two of the omega-3-infused ewes reverted from contractions to nonlabor, an effect never previously observed for experimental glucocorticoid-induced preterm labor in sheep. Maternal plasma estradiol and maternal and fetal prostaglandin E2 rose in control ewes but not in those infused with omega-3 fatty acid. The ability of omega-3 fatty acids to delay premature delivery in sheep indicates their possible use as tocolytics in humans. Premature labor is the major cause of neonatal death and long-term disability, and these studies present information that may lead to a novel therapeutic regimen for the prevention of preterm delivery in human pregnancy.